The functional states and the specific genotype of a cell are predominantly determined by the set of genes expressed in that cell, not only by the number of genes but also by their relative level of expression. In general, both of these critical information can be obtained by an analysis of the level of messenger RNA (mRNA) expressed in the cell. An accurate characterization of the mRNA levels in various cells not only is necessary for a thorough understanding of the fundamental principles governing the determination of each cell type in the body, but can also be used to determine whether a cell has been transformed into a harmful, disease causing state.
Several different methods have been developed to quantify mRNA abundance in biological samples. But, so far, the most reliable, convenient and economical method for the quantitation of mRNA levels is based on the principle of hybridization, which uses target nucleic acid sequences to incubate with a probe sequence under conditions where the complementary probe and its target can form stable hybrid duplexes through base pairing, which is highly sequence specific. By measuring the hybridization results of probes to different mRNA or their cDNA, the expression level of each mRNA species in original sample can be quantified. Blot assays have been used for some time to hybridize nucleic acid material from biological samples to specifically designed probes for analyzing expression level of specific genes in the original sample. Although the traditional blot hybridization assays are well developed, it is not suitable for screening large number of genes and the sensitivity of this technique is relatively low, which requires a large amount of material. This technique further suffers from its low accuracy in quantitative analysis of expression profiles.
The more recent array technology, such as oligonucleotide array or cDNA array, is suited for the analysis of large number of genes, which is made possible by the use of parallel detection of hybridization signals. Both array methods involve physically immobilizing many different hybridization probes on a solid surface in a small array format. Target mRNA or cDNA in solution are hybridized with the tens of thousands different probes defined as “spots” on the surface. The amount of each target species is quantified by scanning individual “spot” in the whole array after targets have hybridized to the probes. This technology has been applied to both basic research and clinical applications. However, a major limitation of this approach is its need for large amount of target material, which is often impossible to obtain. In particular, it has been estimated that even under most ideal conditions, 106–107 of cells would be required for an acceptable signal to noise ratio. The usefulness and the power of this approach notwithstanding, we must recognize that the ability of being able to analyze gene expression at large scale in single or just a few cells is an overwhelming need in both basic research in biology and clinical medicine. In fact, there are many important areas of application where the available materials for analysis cannot be more than a few cells. For example, to understand the principles governing the development of the embryo, we must be able to analyze the first differentiated cells that have a particular spatial relationship. Similarly, to understand how cancer is developed, it is preferable to analyze the early stages of the development, which may or may not exhibit identical traits as mature tumors. Even in the case of biopsy, the small amount of materials available makes it difficult to perform an in-depth analysis of the genes expressed that may be used as reliable markers for tumorigenesis. Therefore, the development of such a powerful technology for both fundamental research and medical diagnosis has constituted a major focus of biotechnology.
Although PCR amplification schemes have been employed to improve upon this lower limit, the large pools of target material generated by amplification have not been shown to faithfully represent the relative abundance of different targets in the original material, especially for those low copy number target species. Apparently, this technology is not applicable to single cell analysis with acceptable reliability. Furthermore, the accuracy of this method is also limited due to detection sensitivity.
A very important point to make, however, is the fact that in most practical applications, there is no need or even the desire to monitor the expression levels of the entire genome which involves hundreds of thousands of genes. Since usually there are up to few hundred genes involved in specific cellular pathways and certain disease such as hypertension, in which it is believed that there are about 150 target genes' expression are modified or changed, information related to these specific groups are of most interests. Another example is the prostate cancer where no more than 500 genes are believed to be involved in this disease.
To date, methods of analyzing gene expressions, which can be applied to single or a limited number of cells, employ fluorescence in situ hybridization, or hybridization based on reverse transcription coupled PCR (RT-PCR) using specific primers. In situ hybridization method has been used to directly visualize the expression of a few particular genes in individual cells, but the procedure involved is rather laborious and the sensitivity of this technique limited its application in gene species. Furthermore, the number of genes that can be analyzed in a sample is very limited. When the later RT-PCR based method is employed with specific primers for amplification, the complexity of the analysis limited the applicable number of genes, which is not realistic for monitoring expression profiles and quantifying the changes in expression levels.
In order to achieve the required sensitivity for single cell analysis and to be able to analyze a large number of genes, a different approach than the current array technology must be pursued. In this application, we describe a practical system and method thereof that is based on sequential detection of hybridization signals that not only is sufficiently sensitive for the analysis of mRNA levels from single cells, but can also allow the quantitation of up to several thousand gene products in a single analytical cycle. Therefore, the applicable range of this technology fulfills the gap between the array technology (suitable for hundreds of thousands of genes by parallel analysis) and the more conventional approaches (only adequate for analyzing small number of gene products). This technology will open the possibility to analyze groups of genes involved in specific pathways, such as development of the embryo and the neuro network, or processes involved in the development of certain diseases, such as hypertension, cancer, with minute amount of materials from few cells and even down to a single cell, as well as applications in clinical medicine, such as the detection of cancerous cells at an early few cell stage, and other genetically determined diseases.